The proposed study will identify, localize and characterize the micro-chemical composition of the initial and subsequent mineral phases in sections of vertebrate bone tissues utilizing the models of developing bone in young rats, chicks, and mice. The research will apply cryo-specimen preparation and electron microdiffraction at 1.2 mega-volts. These are completely new and vital approaches in the study of bone tissues and they will minimize or eliminate artifacts induced in the samples by chemical fixation, staining and electron beam radiation damage incurred during specimen treatment and analysis, respectively. The study should provide information concerning the presence of calcium phosphate mineral phases such as brushite, octacalcium phosphate and/or tricalcium phosphate which have been suggested in bone tissue in addition to the well established hydroxyapatite. The data will be very important in determining the physical chemistry of mineral formation in bone and possible interrelations between putative precursor phases and hydroxyapatite. The localization and distribution of secondary phases in relation to organic extracellular matrix components, cells and cellular ultrastructure will be examined and should provide new information related to the physiological development of bone tissue as a whole. The data compiled using these techniques will be compared and correlated on the basis of structure and physical chemical data obtained by conventional techniques using aqueous fixation and staining of tissue samples. The combination of these techniques represents the most powerful tools available today and should yield the most intimate knowledge of the inorganic constituents of heretofore obtained. The informations that will be obtained are a key to understanding the normal functioning of bone tissue and ultimately to developing concepts and potential solutions to bone tissue pathologies. In addition, in conjunction with other ongoing research in this laboratory examining the role in physiological development of organic components of calcified tissues such as collagenous and non-collagenous extracellular proteins, for example, this new information can provide a better understanding of the role and relationship between inorganic deposits and organic tissue components in bone and vertebrate mineralizing tissues in general.